Calcium (Ca2+) regulates a wide range of functions in the liver in response to hormonal and nutritional signals. The broad goals of this project are to (1) define how nutrients regulate nuclear and cytosolic Ca2+ signaling through post-translational modifications of the inositol 1,4,5 trisphosphate receptor (lnsP3R), a Ca2+ release channel found in both the endoplasmic reticulum (ER) and nucleoplasmic reticulum (NR) and (2) how the dysregulation of this pathway contributes to metabolic liver disease. Nutrient flux leads to posttranslational modifications of cytoplasmic and nuclear proteins by O-linked P-N-acetylglucosamine (OGlcNAc). This dynamic and reversible modification is emerging as a key nutrient sensor and regulator of cell signaling and metabolic physiology. We recently discovered that the lnsP3R is modified by 0-GlcNAc and that this modification decreases lnsP3R single channel activity and Ca2+ release from ER. Preliminary data generated in this PPG suggest that fatty liver induces stress in the ER as well as the nuclear envelope and NR, which may result in impaired cytosolic and nuclear Ca2+ signaling. Based on these findings, we hypothesize that 0-GlcNAcylation of the lnsP3R is controlled by glucose and free fatty acids, which is translated into regulation of the lnsP3R in distinct subcellular compartments of hepatocytes. These regulatory events are, in turn, involved in the perturbation of Ca2+ signaling by ER/NR stress in nonalcoholic fatty liver disease. We will test this hypothesis through the following specific aims: (1) We will identify the effects of glucose and free fatty acids on 0-GlcNAcylation of the lnsP3R isofomns in the nucleus and cytosol; (2) we will examine whether 0-GlcNAcylation of the lnsP3R alters lnsP3-gated channel activity and subsequent intracellular Ca2+ signaling with nuclear and cytoplasmic specificity; and (3) we will detemnine whether metabolic stress promotes hepatic steatosis by perturbing lnsP3R 0-GlcNAcylation and nuclear Ca2+ signaling. Collectively, these studies will synergize with Projects 1 and 3 to define the role of 0-GlcNAcylation of the lnsP3R in nutrient sensing and the regulation of nuclear Ca2+ signaling in the development of hepatic steatosis.